COX-2 (IC50 = 3.9 μM); COX-1 (IC50 = 22.3 μM); FAAH (IC50 = 78.6 μM)

Compound 1, or carprofen, is a non-steroidal anti-inflammatory drug. The IC50 values for COX-2, COX, and FAAH are 3.9 μM, 22.3 μM, and 78.6 μM-1, respectively, as a multi-target inhibitor of FAAH/COX. In CCL and CaCL cells, carprofen (10 μg/mL) has cytoprotective properties and lowers both cell apoptosis. When compared to the corresponding CCL or CaCL controls, PGE2 concentrations were not significantly increased by carprofen (10 μg/mL) [2].
Cytoprotective effects of NSAIDs were dependent on the extent of SNP-induced apoptosis and were greatest in CCL and CaCL cell cultures with moderate SNP-induced cytotoxic effects. Preincubation with an NSAID improved cell viability by 15% to 45% when CCL and CaCL cells were subsequently incubated with SNP. Carprofen (10 μg/mL) had the greatest cytoprotective effects for CCL and CaCL cells. Incubation with NSAIDs resulted in a nonsignificant decrease in PGE(2) production from SNP-damaged cells. Conclusions and clinical relevance: Results indicated that carprofen, meloxicam, and robenacoxib may reduce apoptosis in cells originating from canine cruciate ligaments[2].

On days 3 and 10, carprofen (2.2 mg/kg, po) dramatically lowered the levels of PGE2 in canine blood. On day 3, carprofen similarly decreased the synthesis of PGE2 in the stomach; however, by day 10, the inhibition was not as great. Moreover, on days 3 and 10, it was demonstrated that carprofen had no effect on the synthesis of stomach PGE1 in dogs [3].

In vitro assays[1]
FAAH activity was measured by incubating for 30 minutes at 37°C [3H] anandamide (1 uM cold AEA and 0.6 nM (1 mCi/mL) [3H]-AEA (Arachidonyl-[1-3H] ethanolamine, Specific activity 60 Ci/mmol) in the presence of 50 ug protein/sample of total rat brain homogenates in assay buffer (50 mM TRIS pH 7.4, 0.05 % fatty acid free BSA). The reaction was stopped with cold 1:1 CHCl3/MeOH. The aqueous phase was counted by liquid scintillation (Microbeta2 Lumijet, adapted from Kathuria et al, 2003). Inhibitors were pre-incubated with the enzyme preparation at the appropriate concentration for 10 minutes prior to substrate addition.

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COX activity was measured using a commercial enzyme immunoassay kit. The manufacturer protocol was followed except for the substrate concentration. Briefly, inhibitors were pre-incubated with either ovine COX-1 or human COX-2 for 10 min at 37 °C, and the reaction was carried out in the presence of 5 μM arachidonic acid for 2 minutes at 37 °C. The reaction was stopped with hydrochloric acid and COX-derived PGH2 was then converted to PGF2α with SnCl2. The PGF2α product is then quantified via enzyme immunoassay (EIA) using a PG-specific antibody and competing with a PG-acetylcholinesterase conjugate. Absorbance is measured at 412 nM with a Tecan Infinite M200 plate reader and data processed according to manufacturer’s instructions[1].

Primary cultures of CCL and CaCL cells were created via enzymatic dissociation of cruciate explants. Purified cell cultures were incubated for 2 hours without (controls) or with 1 of 3 concentrations of 1 of 4 NSAIDs (10, 100, or 200 μg of acetylsalicylic acid/mL; 0.1, 1, or 10 μg of carprofen/mL; 0.1, 1, or 10 μg of meloxicam/mL; or 0.1, 1, or 10 μg of robenacoxib/mL) and subsequently incubated for 18 hours with 1 of 3 concentrations of SNP in an attempt to induce mild, moderate, or severe cytotoxic effects. Cell viability and apoptosis were analyzed via a cell proliferation assay and flow cytometry, respectively. Prostaglandin E(2) concentrations were measured via an ELISA[2].

Absorption, Distribution and Excretion
Rapidly and nearly completely absorbed (more than 90% bioavailable) when administered orally.

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Metabolism / Metabolites
Hepatic.

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Biological Half-Life
Approximately 8 hours (range 4.5–9.8 hours) in dogs.

Protein Binding
High (99%)

[1]. Identification and characterization of carprofen as a multitarget fatty acid amide hydrolase/cyclooxygenase inhibitor. J Med Chem. 2012 Oct 25;55(20):8807-26.

[2]. In vitro cytoprotective effects of acetylsalicylic acid, carprofen, meloxicam, or robenacoxib against apoptosis induced by sodium nitroprusside in canine cruciate ligament cells. Am J Vet Res. 2012 Nov;73(11):1752-8.

[3]. In vivo effects of carprofen, deracoxib, and etodolac on prostanoid production in blood, gastric mucosa, and synovial fluid in dogs with chronic osteoarthritis. Am J Vet Res. 2005 May;66(5):812-7.

Carprofen is propanoic acid in which one of the methylene hydrogens is substituted by a 6-chloro-9H-carbazol-2-yl group. A non-steroidal anti-inflammatory drug, it is no longer used in human medicine but is still used for treatment of arthritis in elderly dogs. It has a role as a non-steroidal anti-inflammatory drug, an EC 1.14.99.1 (prostaglandin-endoperoxide synthase) inhibitor and a photosensitizing agent. It is a member of carbazoles and an organochlorine compound.
Carprofen is a non-steroidal anti-inflammatory drug (NSAID) that is used by veterinarians as a supportive treatment for the relief of arthritic symptoms in geriatric dogs. Carprofen was previously used in human medicine for over 10 years (1985-1995). It was generally well tolerated, with the majority of adverse effects being mild, such as gastro-intestinal pain and nausea, similar to those recorded with aspirin and other non-steroidal anti-inflammatory drugs. It is no longer marketed for human usage, after being withdrawn on commercial grounds.
Carprofen is a propionic acid derivate and nonsteroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesic, and antipyretic activities, used exclusively in veterinary medicine. Carprofen inhibits the activity of the enzymes cyclo-oxygenase (COX) I and II, resulting in a decreased formation of precursors of prostaglandins and thromboxanes. This inhibits the formation of prostaglandins, by prostaglandin synthase, that are involved in pain, inflammation and fever. Ibuprofen also causes a decrease in the formation of thromboxane A2 synthesis, by thromboxane synthase, thereby inhibiting platelet aggregation.

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Drug Indication
For use as a pain reliever in the treatment of joint pain and post-surgical pain.

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Mechanism of Action
The mechanism of action of carprofen, like that of other NSAIDs, is believed to be associated with the inhibition of cyclooxygenase activity. Two unique cyclooxygenases have been described in mammals. The constitutive cyclooxygenase, COX-1, synthesizes prostaglandins necessary for normal gastrointestinal and renal function. The inducible cyclooxygenase, COX-2, generates prostaglandins involved in inflammation. Inhibition of COX-1 is thought to be associated with gastrointestinal and renal toxicity while inhibition of COX-2 provides anti-inflammatory activity. In an in vitro study using canine cell cultures, carprofen demonstrated selective inhibition of COX-2 versus COX-1.

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Pharmacodynamics
Carprofen is a non-steroidal anti-inflammatory drug (NSAID) of the propionic acid class that includes ibuprofen, naproxen, and ketoprofen. It is no longer used in the clinical setting, but is approved for use in dogs. Carprofen is non-narcotic and has characteristic analgesic and antipyretic activity approximately equipotent to indomethacin in animal models.

C15H12CLNO2

273.71

273.055

C, 65.82; H, 4.42; Cl, 12.95; N, 5.12; O, 11.69

53716-49-7

Carprofen-d3;1173019-42-5;Carprofen-13C,d3;2012598-34-2

2581

Typically exists as White to off-white solids at room temperature

1.4±0.1 g/cm3

509.1±35.0 °C at 760 mmHg

186-188ºC

261.7±25.9 °C

0.0±1.4 mmHg at 25°C

1.732

4.03

2

2

2

19

362

0

ClC1C([H])=C([H])C2=C(C=1[H])C1C([H])=C([H])C(=C([H])C=1N2[H])C([H])(C(=O)O[H])C([H])([H])[H]

PUXBGTOOZJQSKH-UHFFFAOYSA-N

InChI=1S/C15H12ClNO2/c1-8(15(18)19)9-2-4-11-12-7-10(16)3-5-13(12)17-14(11)6-9/h2-8,17H,1H3,(H,18,19)

2-(6-chloro-9H-carbazol-2-yl)propanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.60 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.60 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.60 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)